In amyotrophic lateral sclerosis (ALS), immune cells and glia contribute to motor neuron (MN) degeneration. We report the presence of NK cells in post-mortem ALS motor cortex and spinal cord tissues, and the expression of NKG2D ligands on MNs. Using a mouse model of familial-ALS, hSOD1 G93A , we demonstrate NK cell accumulation in the motor cortex and spinal cord, with an early CCL2-dependent peak. NK cell depletion reduces the pace of MN degeneration, delays motor impairment and increases survival. This is confirmed in another ALS mouse model, TDP43 A315T . NK cells are neurotoxic to hSOD1 G93A MNs which express NKG2D ligands, while IFNγ produced by NK cells instructs microglia toward an inflammatory phenotype, and impairs FOXP3 + /Treg cell infiltration in the spinal cord of hSOD1 G93A mice. Together, these data suggest a role of NK cells in determining the onset and progression of MN degeneration in ALS, and in modulating Treg recruitment and microglia phenotype.

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Abstract-High blood pressure induces a mechanical stress on vascular walls and evokes oxidative stress and vascular dysfunction. The aim of this study was to characterize the intracellular signaling causing vascular oxidative stress in response to pressure. In carotid arteries subjected to high pressure levels, we observed not only an impaired vasorelaxation, increased superoxide production, and NADPH oxidase activity, but also a concomitant activation of Rac-1, a small G protein. Key Words: high pressure Ⅲ oxidative stress Ⅲ mechanotransduction Ⅲ integrin signaling Ⅲ endothelial dysfunction H ypertension is strictly associated with changes in cardiovascular structure and function that affect morbidity and mortality. 1,2 In particular, at the vascular level hypertension induces an impaired endothelial vasorelaxation, one of the main determinants of cardiovascular risk. 3,4 The increase in pressure within the vasculature generates a biomechanical stress, which is perceived and transmitted to the intracellular compartment through various mechanosensors, and it has been associated with increased production of reactive oxygen species (ROS), which is responsible for vascular dysfunction in hypertension. [5][6][7][8][9] Thus, it could be noteworthy to characterize the intracellular signaling conditioning the cellular machinery toward an increased ROS production in response to biomechanical stress induced by high blood pressure levels. Among the main sources of ROS in the vascular wall, NADPH oxidase seems the most relevant for the vascular dysfunction in hypertension. 7,10 NADPH oxidase is a multisubunit enzyme made up of a membraneassociated catalytic moiety and cytosolic regulatory components that must assemble to form the active oxidase. Activation of NADPH oxidase requires Rac-1, a small G protein, which migrates from cytosol to the plasma membrane, where it favors the assembly of NADPH oxidase subunits. 11,12 Interestingly, an intracellular signaling converging on Rac-1 can be activated not only by agonists binding to G-protein and tyrosine-kinase receptors but also by integrins, 13-15 a class of membrane receptors that link the extracellular matrix to intracellular space. So far, the activation of these latter molecules has been involved in actin polymerization and the rearrangement of the cytoskeleton induced by mechanical forces. 16 -19 Thus, integrins could sense the mechanical force induced by blood pressure on the cell surface and generate an intracellular signaling contributing to the enhanced vascular oxidative stress. 20,21 The aim of this study was to clarify the mechanical stress-induced intracellular signaling toward vascular oxida-

Recent studies described a critical role for microglia in amyotrophic lateral sclerosis (ALS), where these CNS-resident immune cells participate in the establishment of an inflammatory microenvironment that contributes to motor neuron degeneration. Understanding the mechanisms leading to microglia activation in ALS could help to identify specific molecular pathways which could be targeted to reduce or delay motor neuron degeneration and muscle paralysis in patients. The intermediate-conductance calcium-activated potassium channel KCa3.1 has been reported to modulate the "pro-inflammatory" phenotype of microglia in different pathological conditions. We here investigated the effects of blocking KCa3.1 activity in the hSOD1ALS mouse model, which recapitulates many features of the human disease. We report that treatment of hSOD1 mice with a selective KCa3.1 inhibitor, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), attenuates the "pro-inflammatory" phenotype of microglia in the spinal cord, reduces motor neuron death, delays onset of muscle weakness, and increases survival. Specifically, inhibition of KCa3.1 channels slowed muscle denervation, decreased the expression of the fetal acetylcholine receptor γ subunit and reduced neuromuscular junction damage. Taken together, these results demonstrate a key role for KCa3.1 in driving a pro-inflammatory microglia phenotype in ALS.